The objective of the research proposed in this project is to test the hypothesis that DNA organization imposed by DNA-nuclear matrix (NM) attachment points is a key influence in cellular response to radiation-induced DNA damage. To test this hypothesis we propose the following. First, we will identify changes in specific proteins which constitute the NM of cell lines of differing radiosensitivity by 2D-PAGE, microsequencing, intracellular localization and DNA binding capacity. Second, we will characterize the differences between radioresistant and radiosensitive cell lines at the genetic level by differential mRNA display followed y PCR-mediated amplification of radioresistance-associated sequences. Third, we will test models, based on current data which predict differences in the efficiency of repair of double-strand DNA breaks. Testing these models will involve measuring the reparability of DNA double-strand breaks as a function of transciptional status within a DNA domain containing breaks and adjacent domains separated by characterized NM attachment points. Experiments will utilize endonuclease-induced double-stand breaks in specific genes to assess the effects of transcription on the repair of these breaks. Fourth, we propose to develop improved methods to rapidly assay DNA damage in the context of nuclear structure reflecting the observed differences between radiosensitive and radioresistant cells that are applicable to clinical tumor samples so that the above principles can be tested in human tumor cells. Insights gained by accomplishing these specific aims will contribute to an enhanced understanding of intrinsic cellular radiosensitivity and its relationship to tumor radiocurability.